Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) cause millions of lifelong, recurring infections. In adults, HSV infection typically leads to epithelial lesions, which recur whenever the virus reactivates from its latent reservoir in neurons. HSV infection of neonates can lead to more devastating outcomes than those seen in adults, including neurologic impairment or death. Around 1 in 3,200 births results in neonatal HSV disease, often from mothers who are not aware of their infection or of active viral shedding. Half of these neonates experience disseminated multi-organ or invasive disease in the central nervous system (CNS), while the remainder have more limited infections of the skin, eyes, or mouth. Rates of mortality and lifelong morbidity are significantly higher for the invasive CNS and disseminated forms of neonatal infection, than for the more limited surface infections. The contribution of HSV genetic variation to these different clinical outcomes is unknown. However, the genetic dissection of other viruses such as influenza and HIV has led to the identification of viral genetic factors that influence virulence and disease. We therefore propose to conduct a genomic and phenotypic analysis of HSV variation in neonatal infections. We will examine HSV genomic variation using isolates from published clinical studies of neonatal disease outcomes. The combination of viral comparative genomics, phenotypic analyses of cell-to-cell spread, and detailed clinical data will enable us to lay the foundation for a future genome-wide association study (GWAS) for neonatal HSV, which would test for correlations between viral genetic variation and clinical data such as invasive CNS vs. skin disease, severity of neurologic impairment, or response to antiviral chemotherapy. In Aim 1, we will use high-throughput sequencing and viral comparative genomics to dissect all of the viral genetic variations found in ?30 cases of neonatal HSV disease. This will enable us to detect single nucleotide variations (SNPs), insertions, and deletions that correlate with clinical measures of disease, or with in vitro data from Aim 2. In Aim 2 we will use a panel of cellular and biochemical assays to characterize spread phenotypes, protein expression, and antiviral drug resistance for each neonatal HSV isolate. This will reveal any strains with overt growth differences, and enable us to measure the expression and localization of viral proteins that vary in our genomic comparisons. Data on in vitro acyclovir resistance will be compared to clinical data on subsequent recurrences for each patient. These data will establish the foundation for future studies that test hypotheses about how viral genetic variations impact neonatal disease, using animal models of neonatal infection. The identification of viral genetic loci associated with invasive neonatal HSV disease would provide new insights on host-pathogen interactions and potential targets for therapeutic development, as well as informing treatment duration and the use of drugs in development.